Composition and method for delivery of BMP-2 amplifier/co-activator for enhancement of osteogenesis

ABSTRACT

A composition comprising a synthetic growth factor analogue comprising a non-growth factor heparin binding region, a linker and a sequence that binds specifically to a cell surface receptor and an osteoconductive material where the synthetic growth factor analogue is attached to and can be released from the osteoconductive material and is an amplifier/co-activator of osteoinduction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/186,165 filed Jul. 19, 2011 (now U.S. Pat. No. 8,796,212), which is adivision of U.S. patent application Ser. No. 11/767,391 filed Jun. 22,2007 (now U.S. Pat. No. 7,981,862), which claims priority to and thebenefit of U.S. Provisional Patent Application Ser. No. 60/805,594 filedJun. 22, 2006, and the specification thereof of each is incorporatedherein by reference.

BACKGROUND

The present invention relates to compositions that result in enhancedosteogenesis across a broad range of bony repair indications and methodsof using the compositions in a delivery vehicle for improved repair ofbony lesions.

The US published application 20050196425 to Zamora et al entitled,“Positive modulator of BMP-2” teaches a compound comprising a bonemorphogenic protein-2 (BMP-2) analogue which is useful to repair bonelesions and a method in which the compound can augment endogenous orexogenously added BMP-2 activity. It further teaches that there are anumber of commercially available bone graft substitutes that areosteoconductive that the BMP-2 modulator compounds could modify. Theosteoconductive materials included a number of calcium phosphatecontaining composites. The compound is an additive to bone matrix orbone graft materials or controlled or associated with drug deliverydevices among others. 20050196425 however, does not disclose peptide andosteoconductive formulations that permit efficient peptide binding toosteoconductive materials, controlled differential release throughmanipulating the osteoconductive composition, manipulating the peptidecomposition, concentration of the compound attached thereto and/ormanipulating the calcium sulfate concentration. For this application,the positive modulator of BMP-2 will be referred to as theco-activator/amplifier.

Osteoconduction can be described as the process of forming bone on agraft material that is placed into a void in a bony environment. Broadlyspeaking osteoconduction means that bone grows on a surface.Osteoconduction requires a scaffold for cells to move into the graftsite and produce bone. Scaffold materials can be categorized into fourtypes: allograft bone, natural polymers (hyaluronates, fibrin,carboxymethyl cellulose, chitosan, collagen, etc.), synthetic polymers(polylactic acid (PLA), polyglycolic acid (PGA)), and inorganicmaterials (e.g. hydroxyapatite (HA), tricalcium phosphate (TCP), calciumsulfate (CaS)). A number of synthetic osteoconductive bone graftmaterials have been developed for purposes of filling boney voids. Thesegraft materials, however, only osteoconductive and providing a scaffoldfor viable bone healing including ingrowth of neovasculature and theinfiltration of osteogenic precursor cells into the graft site.

Osteoinduction is the process by which osteogenesis is induced and is aprocess regularly seen in any type of bone healing. Osteoinductionimplies the recruitment of immature cells and the stimulation of thesecells to develop into preosteoblasts. In a bone healing environment, themajority of bone healing is dependent on osteoinduction. This process istypically associated with the presence of bone growth factors(principally bone morphogenic proteins) within the bone healingenvironment.

Osteoinduction can be influenced by a number of proteins or growthfactors, growth or new blood vessels (angiogenesis). These proteinscause healing bone to vascularize, mineralize, and functionmechanically. They can induce mesenchymal-derived cells to differentiateinto bone cells. The proteins that enhance bone healing include the bonemorphogenetic proteins, insulin-like growth factors, transforming growthfactors, platelet derived growth factor, and fibroblast growth factoramong others. The most well known of these proteins are the BMPs whichinduce mesenchymal cells to differentiate into bone cells. Otherproteins influence bone healing in different ways. Transforming growthfactor and fibroblast growth factor regulate angiogenesis and caninfluence bone formation and extracellular matrix synthesis.Extracellular matrix molecules such as osteonectin, fibronectin,osteonectin, laminin, and osteocalcin promote cell activation, cellattachment and facilitate cell migration.

While any healing bone lesion is an osteoinductive environment not allosteoinductive environments (bone lesions) have the ability to undergo afull or complete healing. This has lead to the use of recombinant bonemorphogenic proteins to induce osteoinduction in graft materials therebyto induce stem cells to differentiate into mature bone cells.

U.S. Pat. No. 7,041,641 to Rueger et al., demonstrates any number ofbone morphogenic proteins (BMPs) and growth factors combined with anumber of scaffolds (including HA and TCP) and a binder for bone repair.These graft materials are, however, expensive and can lead to exuberantor ectopic bone production.

U.S. Pat. No. 6,949,251 Dalal et al., discloses a beta TricalciumPhosphate (βTCP) particle with any number of BMPs and/or a binder (CMC,Hyaluronate, etc.) for bone repair.

U.S. Pat. No. 6,426,332 Rueger et al., discloses βTCP as anosteoconductive material with any number of bioactive agents combinedtherewith, for example BMP-2. The bioactive agent is dispersed in abiocompatible, nonrigid amorphous carrier having no defined surfaces,wherein said carrier is selected from the group consisting ofpoloxamers; gelatins; polyethylene glycols (PEG); dextrans; andvegetable oils.

A commercially available product for periodontal bone repair, GEM-21S™,utilizes a β-TCP granule coated with platelet derived growth factor“PDGF.” Saito et al., (JBMR 77A:700-6 (2006)) utilized the 73-92 peptidederived from 73-92 of the BMP-2 knuckle epitope. This peptide was coatedon αTCP(OCTCP) cylinders and implanted in 20 mm long defects. Konishi etal., (J. Spine Disorders & Tech.) and Minamide et al., (Spine 200126(8):933-9) demonstrated BMP combined with hydroxyapatite granules forlumbar fusion.

Delivery of small molecules (such as peptides) for therapeuticindications is usually accomplished by various encapsulationtechnologies—microspheres, for example, in which the molecule isencapsulated in a vesicle which degrades over time to release thepeptide. Delivery of a small molecule from the surface of a medicaldevice has been challenging as small molecules rarely have physicalproperties that provide sufficient binding properties to a biomaterialsurface. Often, the peptide is covalently attached to the surface in aneffort to prevent rapid release (Saito et al., J. Biomed Mater Res70A:115-121 (2004; Seol Y-J et al., J. Biomed. Mater Res (A) (2006))(Varkey et al., Expert Opin Drug Deliv. 2004 November; 1(1):19-36.Growth factor delivery for bone repair, Varkey et al.,). One drawback ofcovalent crosslinks is the molecule is unable to release and influencethe surrounding osteoconductive environment.

The delivery kinetics and quantities of a synthetic compound comprisinga BMP-2 amplifier/co-activator may be specifically tailored to theindication of choice. It should be recognized that after a bony lesionis made, there is a reparative response that results in the cellularproduction of BMP-2, and furthermore, that this production occurs over agiven time sequence with an upregulation period eventually followed bydownregulation. Niikura et al., (2006 ORS, #1673) measured BMP-2production over time in standard fractures and non-unions in rats anddemonstrated less BMP-2 production in non-unions than in standardfractures and increasing amounts of BMP-2 up to 21 days followed by adecline in expression at 28 days. BMP-2 expression has been detected inthe human fracture callus (Kloen et al., 2003, 362-371). Furthermore,Murnaghan et al., (JOR 2005, 23:625-631) demonstrated in a mousefracture trial that BMP-2 administered to the fracture at day 0 or 4produced greater repair than that introduced at day 8. It should benoted that in this case BMP-2 is timed with the production of stem cellsthat can be differentiated to bone and is not timed with endogenousBMP-2 production.

A synthetic growth factor identified as B2A2-K-NS was first disclosed byZamora et al in U.S. patent application titled Positive Modulator ofBone Morphogenic Protein-2 having Ser. No. 11/064,039 filed Feb. 22,2005 in addition to disclosing various other peptides. However it wasnot disclosed to combine the synthetic growth factor with anosteoconductive material as a composition for treating bone lesions.

There is, therefore, a need for a composition which can act as a bonevoid filler material and which is comprised of a synthetic growth factoranalogue which can act as an amplifier/co-activator of osteoinduction,and which can attached to and released from an osteoconductive materialto enhance boney repair and healing processes.

There are also number of surgical procedures in orthopedics whereinaugmentation of bone repair would be particularly beneficial includingfusion procedures including those of the spine and ankle; in filling thevoids in bones resultant from traumatic injury; in the treatment ofnon-unions; fracture healing in all skeletal elements; in fixation ofinternal hardware such as rods, plates, screws, and the like; in concertwith spinal cages or vertebral body replacements; and in theaugmentation of implanted wedges, pedicle screws, or rings. These typesof procedures and the associated hardware would be known to thoseskilled in the art.

SUMMARY OF THE INVENTION

According to one aspect, the present invention describes compositionsthat result in enhanced osteogenesis across a broad range of bony repairindications and wherein a synthetic growth factor analogue attached toand released from an osteoconductive material acts as anamplifier/co-activator of osteoinduction and results in enhanced boneyrepair and healing processes. Alternatively, the synthetic growth factoranalogue is affixed to the osteoconductive material and is not released.

According to another aspect, the present invention provides a deliveryvehicle containing the following components: an osteoconductive scaffoldand a synthetic growth factor analogue acting as anamplifier/co-activator of osteoinduction; wherein the scaffold iscapable of binding and releasing the synthetic growth factor, andpreferably at a rate that coincides with the presence of endogenousBMP-2.

In another aspect of the invention the correct release parameters of thesynthetic growth factor analogue is related to the type of bone lesionand results in enhanced osteogenesis. The delivery kinetics andquantities of a synthetic growth factor analogue may be specificallytailored to the indication of choice. For example if the syntheticgrowth factor analogue is intended to augment the activity of endogenousBMP-2, delivery characteristics in a fracture repair should require amore rapid delivery as compared to a spine fusion in which a much slowerdelivery over a much longer period would likely be preferred.

In that regard, it should be recognized that after a bony lesion ismade, there is a reparative response that results in the cellularproduction of BMP-2, and, furthermore, that this production occurs overa given time. Furthermore, it should be recognized that the quantity ofendogenous BMP-2 produced is dependent upon many factors including thesurface area of injured bony tissue, the number of viable osteoblastcells, the rate of repair, etc. Non-critical size defects havesufficient reparative cells and BMP-2 to repair the defect without anexogenous biomaterial. Furthermore, small, segmental fractures mayproduce a much greater amount of host BMP-2 relative to the defectvolume than larger defects that have less bony surface area.

According to yet another aspect, the present invention provides acomposition comprising a synthetic growth factor peptide analoguecomprising a non-growth factor heparin binding region, a liker and asequence that binds specifically to a cell surface receptor; and anosteoconductive material comprising one or more of an inorganicmaterial, a synthetic polymer, a natural polymer, an allograft bone, orcombination thereof, wherein the synthetic growth factor analogue isattached to and can be released from the osteoconductive material and isan amplifier/co-activator of osteoinduction.

The composition of formula II wherein the synthetic growth factoranalogue has the following structure:

wherein:

X is a peptide chain that (i) has a minimum of three amino acidresidues. (ii) has a maximum of about fifty amino acid residues, and(iii) binds specifically to a cell surface receptor;

R₁ is independently hydrogen, such that the terminal group is NH₂, anacyl group with a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl,alkene, alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, orNH group or a corresponding acylated derivative, or is amino acid, adipeptide or a tripeptide with an N-terminus NH₂, NH₃ ⁺, or NH group:

R₆ is independently a linker comprising a chain from 0 to about 15backbone atoms covalently bonded to R₅ when the linker is greater than 0atoms:

R₅ is a trifunctional alpha amino acid residue, wherein X is covalentlybonded through a side chain of R₆;

R₄ is OH such that the terminal group is a carboxyl, NH₂, an acyl groupwith a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl, alkene,alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, or NH groupor a corresponding acylated derivative, or NH—R₁;

Y is a linker comprising a chain from 0 to about 50 backbone atomscovalently bonded to R₅ and Z; and

Z is a non-signaling peptide chain that includes a heparin bindingdomain comprising an amino acid sequence that comprises (i) a minimum ofone heparin binding motif, (ii) a maximum of about ten heparin bindingmotifs, and (iii) a maximum of about thirty amino acids; and anosteoconductive material comprising one or more of an inorganicmaterial, synthetic polymer, natural polymers, allograft bone, orcombination thereof. In a preferred embodiment, the synthetic growthfactor analogue is attached to and can be released from theosteoconductive material and is an amplifier/co-activator ofosteoinduction.

In another aspect of the invention the synthetic growth factor analoguethat is attached to and released from an osteoconductive material is thepeptide B2A2-K-NS. B2A2-K-NS acts as an amplifier/co-activator of BMP-2and via that processes amplifies osteoinduction and results in enhancedboney repair and healing processes. B2A2-K-NS is of the followingsequence: AISMLYLDENEKVVLKK(AISMLYLDENEKVVLK)HxHxHxRKRLDRIARNH₂

B2A2-K-NS binds to inorganic granules including 100% hydroxyapatite (HA)and biphasic compositions of HA. For example, 20:80 (HA:TCP) and 60:40(HA:TCP) but not limited thereto. B2A2-K-NS also binds to organicmaterial (for example, collagen sponge). B2A2-K-NS is released atdifferent rates from several inorganic granules. The magnitude ofpeptide release is altered by peptide concentration and/or by thepeptide amino acid composition. For example, widely distributed positivecharges on the peptide results in less release (e.g. more tightly boundpeptide) than peptide that lacked broad positive charge distribution.Importantly, a rabbit spine fusion study demonstrated that B2A2-K-NSbound to and released from a 20:80 (HA:TCP) granule and resulted inoptimal release characteristics that enhanced endogenous BMP-2 activity,which resulted in enhanced bone formation and spine fusion.

In another aspect of the invention the synthetic growth factor analoguethat is attached to and released from an osteoconductive materialco-activators or amplifies or modifies a biological process such asblood vessel formation, inflammation, cell growth, cell binding toosteoconductive scaffold or chemotaxis that is related to boneformation. Similarly, others of formulas I and II which includeembodiments wherein the X region is all or a portion, or a homolog ofall or a portion of SEQ ID NOs 7-19 but not limited thereto.

Additionally, the following synthetic growth factor analogues may so beused: B7A with the sequence as follows:VLYFDDSSNVILKKK(VLYFDDSSNVILKK)HxHxHxRKRKLERIAR-amide wherein Hx isaminohexanoic acid. B7A enhances BMP-2 activity by increasing activityof BMP-7 and BMP-2.

LA-2 which stimulates cell adhesion and migration may also be used andhas the sequence as follows:SIKVAVAAK(H-SIKVAVAA)HxHxHxRKRKLERIAR-amide. Increasing the number ofcells that bind to an osteoconductive scaffold would indirectly enhancethe activity of endogenous BMP-2.

Also, F2A4-K-NS which induces blood vessel growth can be used to enhancebone formation. F2A is a peptide mimetic of basic FGF, and is alsoreferred to as F2A. Both that peptide and bFGF have been previouslydemonstrated to enhance angiogenesis. Increasing angiogenesis has beenpreviously demonstrated to enhance bone formation and thus would beexpected to increase the bone formation activity of BMP-2 in concert.F2A4-K-NS has the sequence:YRSRKYSSWYVALKRK(H-YRSRKYSSWYVALKR)HxHxHxRKRLDRIAR-amide.

Similarly, the synthetic growth factor analogue VA5, which is a mimeticof vascular endothelial growth factor may so be used and has thefollowing sequence: WFLLTMAAK(WFLLTMAA)HxHxHxRKRKLERIAR-amide.

Also, the synthetic growth factor analogue SD-2 which mimics aspects ofstromal derived growth factor-1 may be used to increase chemotaxis andlocalization of circulating progenitor cells to the bone lesion site.SD-2 has the sequence: KWIQEYLEKK(KWIQEYLEK)HxHxHxRKHxRKLERIAR-amide.

This invention can also utilize other heparin binding growth factoranalogues based on vascular endothelial growth factor which increasecell growth and be related to platelet derived growth factor ortransforming growth factor-beta and the like which would act in accordwith this invention to enhance osteoinduction and accelerate bonerepair.

Similarly, synthetic growth factor analogues which bind directly to theBMP-2 or its receptor, generally similar to B2A2-K-NS described hereincan also be used to amplify biological processes that increase boneformation. Release of these peptide would occur over the correct time soas to optimize this related biological process.

In another aspect of the invention the composition of this invention maybe used with exogenously supplied osteoinductive agents. Theseosteoinductive agents can include demineralized bone matrix other formof allograft material.

In another aspect of the invention the composition of this invention maybe used with exogenously supplied osteoinductive agents based onrecombinant technologies. These recombinant osteoinductive agentsinclude BMP-2, BMP-7 (OP-1), GDF-5 (MP-52), TGF-beta1 and others thatare known to those skilled in the art.

In another aspect of the invention the composition of this invention maybe used with autograft bone or bone marrow aspirate that is added withthe bone replacement graft at the lesion site.

Additional objects and advantages of the present invention will beapparent in the following detailed description read in conjunction withthe accompanying drawing figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a graph of B2A2-K-NS release from inorganic granulesaccording to one embodiment of the present invention.

FIG. 2 illustrates B2A2-K-NS release from different osteoconductivematerials.

FIG. 3 illustrates B2A2-K-NS release over 28 days according to oneembodiment of the present invention.

FIG. 4 illustrates B2A2-K-NS release at several concentrations from anosteoconductive material according to one embodiment of the presentinvention.

FIG. 5 illustrates three different peptides released from anosteoconductive material according to one embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions: As used here and elsewhere, the following terms have themeanings given.

The term “a” as used herein means one or more.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of such alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The “alkyl” groups specified herein include those alkyl radicals of thedesignated length in either a straight or branched configuration.Examples of such alkyl radicals include methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,isohexyl, and the like.

The term “aryl” includes a monovalent or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkythio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. Examples of an aryl group include phenyl, biphenyl,naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like.

The term “aralkyl” includes a radical —R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like. The term “aliphatic”includes compounds with hydrocarbon chains, such as for example alkanes,alkenes, alkynes, and derivatives thereof.

The term “acyl” includes a group RCO—, where R is an organic group. Anexample is the acetyl group CH₃CO—.

A peptide or aliphatic moiety is “acylated” when an alkyl or substitutedalkyl group as defined above is bonded through one or more carbonyl{—(C═O)—}groups. A peptide is most usually acylated at the N-terminus.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—CO.NH₂).

An “amine” includes compounds that contain an amino group (—NH₂).

A “diamine amino acid” is an amino acid or residue containing tworeactive amine groups and a reactive carboxyl group. Representativeexamples include 2,3 diamino propionyl amino acid, 2,4 diamino butylicamino acid, lysine or ornithine.

The term “Hx” as used herein means aminohexanoic acid and is alsosometimes abbreviated Ahx.

The term “homologous”, as used herein refers to peptides that differ inamino acid sequence at one or more amino acid positions when thesequences are aligned. For example, the amino acid sequences of twohomologous peptides can differ only by one amino acid residue within thealigned amino acid sequences of five to ten amino acids. Alternatively,two homologous peptides of ten to fifteen amino acids can differ by nomore than two amino acid residues when aligned. In another alternative,two homologous peptides of fifteen to twenty or more amino acids candiffer by up to three amino acid residues when aligned. For longerpeptides, homologous peptides can differ by up to approximately 5%, 10%,20%, 25% of the amino acid residues when the amino acid sequences of thetwo peptide homolog are aligned.

A “trifunction amino acid is an amino acid is an amino acid or residuewith three reactive groups, one the N-terminal amine, a second theC-terminus carboxyl, and the third comprising all or a part of the sidechain. Trifunctional amino acids thus include, by way of example only,diamine amino acids; amino acids with a reactive sulfhydryl group in theside chain, such as mercapto amino acids including cysteine,penicillamine, or 3-mercapto phyenylalanine; amino acids with a reactive

The term “synthetic growth factor analogue” as used herein may be offormula I or II. Each synthetic growth factor analogue of the inventioncontains two substantially similar sequences (homodimeric sequences) atX that are analogues of a particular growth factor that binds to agrowth factor receptor that may be located on the cell surface, oralternatively that bind to a growth factor receptor without being ananalogue of the cognate growth factor. The homodimeric sequences may bederived from any portion of a growth factor. The synthetic GROWTH FACTORanalogue may be an analogue of a hormone, a cytokine, a lymphokine, achemokine or an interleukin, and may bind to any growth factor receptor,for any of the foregoing.

According to one embodiment of the present invention a composition fortreatment of bone lesions comprises a synthetic growth factor analoguewhich acts as a amplifier/co-activator of endogenous BMP-2 and is offormula I or II, and is releasably attached to an osteoconductivematerial. The compound of formula I

wherein:

X is a peptide chain that (i) has a minimum of three amino acidresidues, (ii) has a maximum of about fifty amino acid residues, and(iii) binds specifically to a specifically to a cell surface receptor;

R₁ is independently hydrogen, such that the terminal group is NH₂, anacyl group with a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl,alkene, alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, orNH group or a corresponding acylated derivative, or is amino acid, adipeptide or a tripeptide with an N-terminus NH₂, NH₃ ⁺, or NH group;

R₂ is independently a trifunctional alpha amino acid residue, wherein Xis covalently bonded through a side chain of R₂:

R₃ is independently a linker comprising a chain from 0 to about 15backbone atoms covalently bonded to R₂;

R₄ is OH such that the terminal group is a carboxyl, NH₂, an acyl groupwith a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl, alkene,alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, or NH groupor a corresponding acylated derivative, or NH—R₁;

Y is a linker comprising a chain from 0 to about 50 backbone atomscovalently bonded to R₂ and Z; and

Z is a non-signaling peptide chain that includes a heparin bindingdomain comprising an amino acid sequence that comprises (i) a minimum ofone heparin binding motif, (ii) a maximum of about ten heparin bindingmotifs, and (iii) a maximum of about thirty amino acids.

The compound may further comprise a linker that (i) is hydrophobic, (ii)comprises a chain of a minimum of about 9 and a maximum of about 50backbone atoms, and (iii) is not found in Bone Morphogenic Protein-2.The compound may contain at R₂ an L- or D-diamine amino acid residue. Ina preferred embodiment, the L- or D-diamine amino acid residue is 2,3diamino propionyl amino acid, 2,4 diamino butylic amino acid, lysine orornithine.

In one embodiment X is covalently bonded to R₂ and wherein the covalentbonds comprise an amide, disulfide, thioether, Schiff base, reducedSchiff base, imide, secondary amine, carbonyl, urea, hydrazone or oximebond. In a preferred embodiment, X is covalently bonded to R₃ when R₃>0atoms and wherein the covalent bond comprises an amide, disulfide,thioether, Schiff base, reduced Schiff base, imide, secondary amine,carbonyl, urea, hydrazone or oxime bond. Y comprises a straight chainamino carboxylic acid.

The compound of formula II comprising:

wherein:

X is a peptide chain that (i) has a minimum of three amino acidresidues, (ii) has a maximum of about fifty amino acid residues, and(iii) binds specifically to a specifically to a cell surface receptor;

R₁ is independently hydrogen, such that the terminal group is NH₂, anacyl group with a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl,alkene, alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, orNH group or a corresponding acylated derivative, or is amino acid, adipeptide or a tripeptide with an N-terminus NH₂, NH₃ ⁺, or NH group;

R₆ is independently a linker comprising a chain from 0 to about 15backbone atoms covalently bonded to R when the linker is greater than 0atoms;

R₅ is a trifunctional alpha amino acid residue, wherein X is covalentlybonded through a side chain of R₆;

R₄ is OH such that the terminal group is a carboxyl, NH₂, an acyl groupwith a linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl, alkene,alkenyl or aralkyl chain including an N-terminus NH₂, NH₃ ⁺, or NH groupor a corresponding acylated derivative, or NH—R₁;

Y is a linker comprising a chain from 0 to about 50 backbone atomscovalently bonded to R₅ and Z; and

Z is a non-signaling peptide chain that includes a heparin bindingdomain comprising an amino acid sequence that comprises (i) a minimum ofone heparin binding motif, (ii) a maximum of about ten heparin bindingmotifs, and (iii) a maximum of about thirty amino acids.

For either of formula I or II the regions X and Z of the syntheticgrowth factor analogues include amino acid residues, and optionally theregion Y includes amino acid residues. An amino acid residue is definedas —NHRCO—, where R can be hydrogen or any organic group. The aminoacids can be D-amino acids or L-amino acids. Additionally, the aminoacids can be α-amino acids, β-amino acids, γ-amino acids, or δ-aminoacids and so on, depending on the length of the carbon chain of theamino acid.

The amino acids of the X, Y and Z component regions of the syntheticgrowth factor analogues of the invention can include any of the twentyamino acids found naturally in proteins, i.e. alanine (Ala, A), arginine(Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys,C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (H is, H), isoleucine, (Ile, I), leucine (Leu, L), lysine(Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V).

Furthermore, the amino acids of the X, Y and Z component regions of thesynthetic growth factor analogues of the invention can include any ofthe naturally occurring amino acids not found naturally in proteins,e.g. β-alanine, betaine (N,N,N-trimethylglycine), homoserine,homocysteine, γ-amino butyric acid, ornithine, and citrulline.

Additionally, the amino acids of the X, Y and Z component regions of thesynthetic growth factor analogues of the invention can include any ofthe non-biological amino acids, i.e. those not normally found in livingsystems, such as for instance, a straight chain amino carboxylic acidnot found in nature. Examples of straight chain amino carboxylic acidsinclude 6-aminohexanoic acid, 7-aminoheptanoic acid, 9-aminononanoicacid and the like.

For example Z may be selected from one of the following heparin bindingsequences BBBxxB (SEQ ID NO 1 ), BxBB (SEQ ID NO 20 ) where each B isindependently lysine, argininge, ornithine, or histidine and each x isindependently a naturally occurring amino acid. Alternatively, Z may beRKRKLEGIAR (SEQ ID NO 2), RKRKLGRIAR (SEQ ID NO 3 ), RKRKLWRARA (SEQ IDNO 4 ), RKRLDRIAR (SEQ ID NO 5 ), RKRKLERIAR C (SEQ ID NO 6 ).

A synthetic growth factor analogueue of one embodiment of the presentinvention, including those of formulas I and II, provides that the Xregion is all or a portion, or a homolog of all or a portion, of any ofthe following amino acid sequences: AISMLYLDENEKVVL (SEQ ID NO:7),ISMLYLDENEKVVLKNY (SEQ ID NO:8), LYFDESSNVILKK (SEQ ID NO:9),LYVDFSDVGWNDW (SEQ ID NO:10), EKVVLKNYQDMWEG (SEQ ID NO:11),CAISMLYLDENEKVVL (SEQ ID NO:12), AFYCHGECPFPLADHL (SEQ ID NO:13),PFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:14). VLYFDDSSNVILKKK (SEQ ID NO 15 ),SIKVAVAAK (SEQ ID NO 16 ), YRSRKYSSWYVALKRK (SEQ ID NO 17 ), WFLLTMAAK(SEQ ID NO 18 ), or KWIQEYLEKK (SEQ ID NO 19 ). In a preferredembodiment the X region is the amino acid sequence ISMLYLDENEKVVLKNY(SEQ ID NO:8). More preferably the X region is the amino acid sequenceLYFDESSNVILKK (SEQ ID NO:9). More preferably still, the X region is theamino acid sequence AISMLYLDENEKVVL (SEQ ID NO:7).

The osteoconductive material comprises at least one of the compoundsselected from a calcium salt, a collagen, a hydroxyapatite, a ceramic,and also includes demineralized bone matrix or other allograft material.

The osteoconductive material can be formed as a granule, a gel, a putty,a powder, a block or a combination thereof. In a more preferredembodiment, the synthetic BMP-2 compound is B2A2-K-NS.

In another embodiment, the calcium sulfate (CaS) of the osteoconductivematerial is less than about 80 wt % of the material. In a preferredembodiment the CaS is between about 30-80 wt % of the granule.

In yet another embodiment, the osteoconductive material containsinorganic material consisting of about 20-100 wt % HA and about 0-75 wt% TCP. In yet another embodiment, the osteoconductive material comprisesCaS, PGA fibers, and PLG. PGA, PLA and PLG are examples of syntheticpolymers. In still another embodiment of the present invention, theosteoconductive material comprises silicate substituting a portion ofthe phosphate ions in the Hydroxyapatite lattice.

In another embodiment, the osteoconductive material comprises a naturalpolymer like collagen.

In another embodiment, the osteoconductive material comprises one ormore compounds selected from allograft bone, natural polymers orsynthetic polymers.

In yet another embodiment, the osteoconductive materials is formed intogranules, powders, gel, putty or any combination thereof.

In still another embodiment, the osteoconductive material is used incombination with a load bearing device to treat bone lesions.

In a further embodiment, the load bearing device is selected from cages,wedges, rods, pedicle screws, vertebral body replacement, intervertebralbody fusion device, or rings for bone fusion, for example a spinefusion.

In yet another embodiment, the method further comprises adding host bonechips (autograft) in combination with the osteoconductive material nearthe bone lesion. The autograft can be obtained from the iliac crest orfrom the ‘local’ area of surgery. For example, during lumbar spinalfusion surgery, the local bone can be obtained during a facetectomyand/or a laminectomy. The combined heparin binding growth factor coatedon an osteoconductive material is combined with autograft and can bedelivered to the site of spine fusion. The sites can be cervical orlumbar in nature. Furthermore, the surgical approaches can include PLF(posterolateral fusion), PLIF (posterior lumbar interbody fusion), TLIF(transforaminal lumbar interbody fusion), or others that are known tothose skilled in the Art. The methods can furthermore entail posteriorfixation devices such as pedicle screws/rods and others that are knownto those skilled in the Art.

Another embodiment of the present invention comprises a method fortreating bone lesions. The method comprises providing a synthetic BMP-2analogue peptide that is an amplifier/co-activator of endogenous BMP-2to an osteoconductive material such that it is coated or bound to thatmaterial. The osteoconductive material and associated analogue isimplanted in a bone lesion with subsequent release from theosteoconductive material. The active form of the analogue peptideaugments endogenous BMP-2 to treat a bone lesion.

In still another embodiment, releasing the synthetic growth factoranalogue from the osteoconductive material is rate controlled bymanipulating the composition of the osteoconductive material,manipulating the amino acid composition, concentrating the syntheticBMP-2 peptide attached to the osteoconductive material and/orcontrolling the calcium concentration in the osteoconductive material.

Yet another embodiment of the present invention comprises a kit. The kitcomprises a vial of lyophilized vial of synthetic growth factoranalogue, and separately a container of osteoconductive, inorganicgranules. The kit is used to formulate an implantable material byhydrating the analogue with saline or water and mixing the resultantsolution with the granules, whereupon analogue becomes attached to thegranule. After discarding the solution, the granules with the attachedanalogue is then delivered to the bony lesion.

The following table describes heparin binding growth factor analoguesand their biological activity that could promote bone formation whenreleasably attached to an osteoconductive matrix.

TABLE 1 Host Sequence Biological activity associated with Name Relatedto Sequence ID bone formation F2A Basic FGF SEQ ID 17 Angiogenesis B7ABone SEQ ID NO 15 Binding to the BMP-7 receptor; co- Morphogeneticactivation with BMP-2 as a Protein - 7 heterodimer B2A2-K-NS Bone SEQ IDNO 7 Binding to the BMP-2 receptor; co- Morphogenetic activation ofendogenous BMP-2 Protein - 2 SD-2 Stromal Derived SEQ ID NO 19Chemotaxis Factor - 1 LA-2 Laminin SEQ ID NO 16 Increase cellattachment, promote chemotaxis and angiogenesis VA5 Vascular SEQ ID NO18 Increases angiogenesis endothelial growth factor

Embodiments of the present invention are further illustrated with thefollowing examples.

EXAMPLES Example 1 Comparative Binding of B2A2-K-NS to OsteoconductiveBone Replacement Graft from Different Sources

An evaluation of several commercially available osteoconductive bonereplacement grafts (BRGs) was performed to determine their binding andrelease characteristics of the BMP-2 amplifier/co-activator—known asB2A2-K-NS.

First, the materials were placed into 0.9% saline and the pH recorded.

The next step was to determine whether the peptide was able to bindthese BRGs. B2A2-K-NS (approximately 20 ug/ml) was incubated in one mlof 0.9% saline solution with 0.17 gram BRGs for 15 minutes. An ELISAassay was performed both on peptide solution without BRGs and thesupernatant from the peptide+BRG. The difference between the peptideonly reading and the supernatant from the peptide+BRG provided theamount of peptide bound.

The third step was to measure the amount of peptide released using thesame ELISA assay. Briefly, the peptide bound BRGs were placed in 1 ml of0.9% saline and the amount of peptide released was measured over a 72hours period.

The data is summarized in the table below.

TABLE 2 Level of peptide Composition pH binding Peptide Released? 100%HA 6.6 High N/D 100% HA with 10.0 Intermediate Yes silicon substitutions75:25 7.5 Intermediate N/D (HA:TCP) 65:35 7.3 None N/A (HA:TCP) 60:40(HA:TCP) 8.8 High Yes 60:40 (HA:TCP) 6.3 High Yes 20:80 6.5 High Yes(HA:TCP) 100% b-TCP 7.0 None N/A Triphasic: 9.0 High Yes HA, TCP, CaS(1% w/w) Triphasic: 10.5 High No HA, TCP, CaS (10% w/w) PGA fibers + 3.7High No ~9% CaS + PLG

The binding definitions are defined as follows: None is no detectablebinding. High is binding >20 ug peptide/cc graft material. Intermediateis less than 20 ug peptide/cc graft material, but above non detectablebinding. B2A2-K-N-S was found to bind to BRGs in the pH range of3.7-10.5.

B2A2-K-NS was found to bind HA or HA/TCP containing BRGs ranging from100% to 20% hydroxyapatite and from 80% to 25% tricalcium phosphate.

B2A2-K-NS was found to bind HA/TCP/CaS BRG ranging from 0% to 10%calcium sulfate. B2A2-K-NS was found to bind a BRG that containspolyglycolic and polylactic acid combined with calcium sulfate.

A surprise finding was that materials that are similar in compositiondiffer greatly in their ability to bind B2A2-K-NS. Whereas two 60:40HA:TCP and one 75:25 HA:TCP BRGs were able to bind B2A2-K-NS, a 65:35HA:TCP composition was unable to bind B2A2-K-NS.

The relative amounts of HA:TCP did not determine whether the peptide wasreleased or not. For example, one 60:40 HA:TCP BRG released the peptideat ‘high’ rates whereas the other 60:40 HA:TCP BRG released the peptideat ‘intermediate’ rates. More importantly for release was the presenceand quantity of CaS in the granule. B2A2-K-NS could not release from thebiomaterial at acceptable levels at a CaS amount of ˜9-10%, but couldrelease at a CaS concentration of 1%.

B2A2-K-NS could release from the biomaterial at acceptable levels frommaterials ranging from 75% to 20% hydroxyapatite and 80% to 25%tricalcium phosphate. The peptide could release from materials in the pHrange of 6.3-10.

Example 2 Binding of B2A2-K-NS to Ceramic Granules

Methods. 1.5 cc of granules were added to 1.8 mL of 0.9% saline or ofpeptide solution in 0.9% saline having a concentration of 35.9, 119.8,359.3 μg/mL. The granule-peptide solutions were swirled vigorously for15 seconds every 5 minutes for 20 minutes. After swirling, thesupernatant was removed, filtered through 0.22 μm filter (low proteinbinding durapore (PVDF)) and assayed via Bicinchoninic Acid (BCA) ormicro BCA assay (Pierce, Rockport, Ill.) for peptide content. The amountof peptide bound to the particles was obtained by subtraction method(the total peptide minus peptide present in the supernatant is equal tothe amount of peptide bound on particulate materials.). The study wasperformed on two separate days and the results averaged below:

TABLE 3 B2A2-K-NS concentration B2A2-K-NS bound Granule (ug/ml) (%)60:40^((pH 8.8)) 36 85 (HA:TCP) 60:40^((pH 8.8)) 120 90 (HA:TCP)60:40^((pH 8.8)) 359 94 (HA:TCP) 20:80 36 67 (HA:TCP) 20:80 120 88(HA:TCP) 20:80 359 95 (HA:TCP) 100% HA + silicon 359 73 100% HA 230 87

The study demonstrates that a high percentage of B2A2-K-NS peptide isbound on materials that include 100% hydroxyaptite and biphasiccompositions comprised of 20:80 and 60:40 (HA:TCP).

Example 3 Release of B2A2-K-NS from Ceramic Granules

Methods: After binding the peptide to the granules as described inExample 2, each of the vials containing particulate materials wasreplenished with 1.8 mL of 0.9% saline and placed on the rockingplatform. After specified time interval, the supernatant was removed,filtered, and collected over a period of 43 hrs. Based on the amount ofthe peptide present in the supernatant, the amount of peptide bound toor released from the particles as was calculated. The study wasperformed on two separate days with the averages plotted showingcumulative peptide released vs. time (see graph below). (Note: the HAplus silicon was only performed at high dose). A BCA assay was utilizedto determine peptide concentrations. The 60:40 granule was pH 8.8.

Referring now to FIG. 1, the release characteristics of specifiedmaterials tested. There was a burst of peptide released over a 4 hourperiod (supernatant was assessed hourly for the first four hours). Athigh loading doses (431 ug peptide/cc granule), the burst phaseaccounted for 12-22% of the total amount of peptide loaded. Forintermediate loading doses (144 ug peptide/cc granule), the burst phaseaccounted for 25-35% of the total amount loaded. For low doses (43 ugpeptide/l cc granule), the burst phase accounted for 48-59% of the totalamount loaded. Following the burst period, there was a slower, sustainedrelease period.

FIG. 1 illustrates similarly shaped B2A2-K-NS peptide release curves forgranules consisting of 100% hydroxyapatite and biphasic compositionscomprising 20:80 and 60:40 HA:TCP (pH 6.3).

The diamond on the graph represents 60% HAP and 40% TCP (high dose). Thesquare represents 60% HAP and 40% TCP (medium dose). The trianglerepresents 20% HAP and 80% TCP (high dose). The X represents 20% HAP and80% TCP (medium dose). The star represents 100% HAP plus Si (high dose).The filled-in circle represents 60% HAP and 40% TCP (low dose) and theplus sign represents 20% HAP and 80% TCP (low dose.)

Referring now to FIG. 2, the study was repeated using similar conditionsas above using the high B2A2-K-NS dose. The granule size was 1-3 mmexcept for Pro-Osteon, which was ˜0.5 mm. The bovine serum albumin (BSA)standard curve was generated using the supernatant from the granules(without peptide). The B2A2-K-NS BCA value was corrected relative to theBSA standard curve.

FIG. 2 illustrates similarly shaped B2A2-K-NS peptide release curves forgranules consisting of 100% hydroxyapatite (Pro-Osteon) and biphasiccompositions comprising 20:80 and 60:40 HA:TCP (MBCP).

To those skilled in the art, it should be recognized that to reproducethese results, the same volume of supernatant and the same number ofcollection points listed above should be utilized. Increasing thesupernatant volume or increasing the number of supernatant collectionsover a given time period will likely increase the observed amount ofpeptide released. Conversely, decreasing the supernatant volume ordecreasing the number of supernatant collections over a given timeperiod will likely decrease the observed amount of peptide released. Inaddition, modification of the supernatant solution from 0.9% saline towater or another solvent will likely alter the total quantity released.Omitting the supernatant filtration will increase the recovered amountof peptide and increasing the swirling rate may increase the amount ofpeptide released. Performing the BSA standard curve with supernatantreleased from the granules may decrease the peptide quantity observed.

Example 4 Efflux of B2A2-K-NS from Ceramic Granules

The experiment described in Example 3 was repeated using 20:80 granulescoated with 300 ug B2A2-K-NS/ml granules. 3 cc granules were used; 3.6ml of 0.9% saline was collected at the time points to determine peptiderelease. Referring now to FIG. 3 a long-term cumulative B2A2-K-NSrelease over a period of 28 days is illustrated. The two curves show theamount of peptide released after filtration (squares) or prior tofiltration (diamonds) of the supernatant. A 0.22 um filter (low proteinbinding durapore PVDF) was utilized.

Example 5 Method Used for Determination of Release of B2A2-K-NS fromGranules

Peptide release from 20% HAP/80% TCP was determined using the followingmethod. 1.5 cc of granules were added to 1.8 mL of 0.9% saline or ofpeptide solution in 0.9% saline having a concentration of 43, 143 or 431μg B2A2-K-NS/cc granule. The granule-peptide solutions were swirledvigorously for 15 seconds every 5 minutes for 20 minutes. Afterswirling, the supernatant was removed and assayed via BCA or micro BCAassay for peptide content.

Referring now to FIG. 4, a similarly shaped release curve is generatedfor the same peptide at high dose (diamonds), medium dose (squares) andlow dose (triangles) and illustrates that the peptide coatingconcentration affects the magnitude of the release.

Example 6 Comparative Binding of Synthetic Growth Factor Analogues toCeramic Granules

In order to better determine the physical characteristics of peptidebinding and release, several peptides were compared on a singlescaffold. Although the other peptides tested were not co-activators ofBMP-2, they share a similar structure in that they are branched, of asimilar size and contain a heparin binding domain.

Methods for assaying peptide release from a single scaffold are asfollows: 1 cc of granules were added to 1.2 mL of 0.9% saline or ofpeptide solution in 0.9% saline having a concentration of 100 μgB2A2-K-NS, LA2 or F2A4/cc granule. The granule-peptide solutions wereswirled vigorously for 15 seconds every 5 minutes for 20 minutes. Afterswirling, the supernatant was removed and assayed via BCA or micro BCAassay for peptide content. The BCA value for each peptide was correctedrelative to the BSA standard curve.

Referring now to FIG. 5, the shape of the release curve is similar forall three peptides (B2A2-K-NS (diamonds), LA2 (squares) and F2A4-K-NS(triangles), however, the magnitude of the release is markedlydifferent. The following table shows the amino acid composition of eachpeptide.

TABLE 4 Hydro- + charges phobic Aro- Length Peptide incl. NH2 − chargesIncl. Ahx matic peptide MW LA2 10 1 18 0 30 3272 B2A2-K-NS 11 7 20 2 455344 F2A4 17 1 12 8 43 5540

The number of positive, negative, or hydrophobic amino acids for eachpeptide were similar and does not appear to account for the largedifferences in the observed release rates. Charge distribution of eachpeptide is illustrated in FIG. 6.

The charge distribution on the amino acid terminus is similar in allpeptides and so this region would not be expected to contribute to thedifferences in release rates. One possible reason for why F2A4 does notrelease readily is the wide distribution of positive amino acids, whichcould interact with the negatively charged hydroxyapatite/TCP surface.The aromatic amino acids may help distribute the positive charges acrossa greater region of the peptide. The number of positive charges is notinterrupted by any negative charges. In contrast, B2A2-K-NS has fewerpositively charged groups and the positive charges are interrupted bynegatively charged groups. The most rapidly released peptide, LA2, onlyhas two amino acids at the very carboxy terminus and these amino acidsare bordered on one side by non-aromatic hydrophobic residues. The longregion of non-aromatic hydrophobic domain located in the branched areaof the peptide may also promote easy dislodging, as neither F2A4 orB2A2-K-NS has such an extensive hydrophobic region.

Example 7 Bioactivity of B2A2-K-NS Released from Ceramic Granules

Methods: (A) Peptide was collected over a six hour period after releasefrom 20:80 (as described in Example 3). The supernatants from all timepoints were mixed, frozen and lyophilized for 48 hours. The peptidepellet was reconstituted in 1.5 ml saline and a BCA assay performed todetermine peptide concentration.

(B) Alkaline phosphatase assays were performed using mouse pluripotentcell lines C2C12. Cells were plated in 96-well (1×10⁴/well) plates inDMEM/F-12 Ham's containing 15% FBS, 1% L-gln, 1% NaPyr, and 1%gentamycin and allowed 24 hours to attach at 37 C., 5% CO₂, 90%humidity. The medium was then aspirated and replaced with a low-serum(5% FBS) medium containing B2A2-K-NS in various concentrations rangedfrom 1.25- to 10 μg/ml, with or without addition of BMP-2 (100 ng/ml).After 1 day, ALP activity was determined.

Results and Conclusion: B2A2-K-NS peptide released from 20:80 increasedBMP-2 activity, which demonstrated B2A2-K-NS activity after release from20:80 granules.

Example 8 Binding of B2A2-K-NS to Collagen

Type I, bovine derived, dermal collagen sheets that were crosslinkedwith DHT and lyophilized, were incubated with 1.072 ug B2A2-K-NS/mlcollagen sheet in 0.9% saline solution. The binding efficiency was 61%,which was far lower than the binding efficiency on the Ca²⁺ containinggranules at high B2A2-K-NS coating concentrations (87-95% as describedin Example 2). Lower binding efficiencies may be attributed to lowersurface area as compared to that of the granules.

To initiate release, a vial containing collagen coated with B2A2-K-NSwas replenished with 2 mL of 0.9% saline and placed on the rockingplatform. After specified time interval, the supernatant was removed,filtered, and collected. A BCA assay was utilized to determine peptideconcentrations. Hourly collections in 0.9% saline demonstrated that thecumulative amount of peptide released was 11, 19, 23, and 28% after 1,2, 3 and 4 hours respectively. The BSA standard curve was generated byplacing BSA in supernatant from the collagen sponge (without peptide).The release rates from collagen were faster than the release rates from20:80 using comparable conditions.

Example 9 Use of B2A2-K-NS/Ceramic Granules to Augment PosteriolateralSpinal Fusion in Rabbits

All procedures were approved by the Institutional Animal Care UseCommittee. Skeletally mature New Zealand White Rabbits weighing 4.5-5.5kg were individually caged and monitored daily for signs of pain anddiscomfort. All operative procedures were performed in a surgical suiteusing inhalation anesthesia and aseptic techniques. A pre-anestheticdose of Ketamine HCL 26 mg/Kg, Acepromazine Maleate 0.15 mg/Kg, andXylazine HCL 0.78 mg/Kg was administered IM. Rabbits were masked and1.5-2.5% isoflurane was delivered in O₂.

A single level posterolateral lumbar intertransverse process fusion(PLF) was performed in 60 rabbits, bilaterally at L4-L5, with autogenousbone graft from the iliac crest and differing doses of B2A2-K-NS.Rabbits were placed prone on the operating table and surgically preppedwith 70% Betadine solution. A dorsal midline incision, approximately 15centimeters long, was made from L1 to the sacrum and the soft-tissuesoverlying the transverse processes (TP) were dissected. The TPs werethen decorticated with a high-speed burr.

The study consisted of the following materials (10 animals pertreatment) placed between the transverse processes in the paraspinal bed(3 ml per side): the autograft group consisting of morselized,cancellous bone graft; the open group (no graft material): the granuleonly group (no B2A2-K-NS coating concentration) (B2A2-K-NS/G 0 ug); thelow coating concentration treated granule group (B2A2-K-NS/G 50 ug), themedium coating concentration treated granule group (B2A2-K-NS/G 100 ug)and the high coating concentration treated granule group (B2A2-K-NS/G300 ug). All animals treated with granules contained approximately 50%autograft by volume. The granule used was 20:80 (HA:TCP).

Fascia and skin were closed with 3-0 Vicryl and then the skin wasstapled. Cephalothin (13 mg/kg) was administered prior to surgery andtwice a day for 5 days postoperatively. To insure the animals werecomfortable, analgesics are administered based on the observation of thePI. Butorphanol (1-7.5 mg/kg IM q4) and Flunixin Meglumine (1.1 mg/kg IMq12) were given daily for 48 hours post-op. Euthanasia was performed atsix weeks followed by radiographs and manual palpation by three blindedexaminers to determine fusion. If one side was deemed fused by two outof three examiners then the spine was determined fused. All surgeonswere also blinded to the treatment at the time of surgery. Fusion wasalso assessed via examination of radiographic plain films.

TABLE 5 Manual Palpation Radiographic Treatment fusion rate (%) fusionrate (%) Open 0 0 Autograft - positive control 63 55 B2A2-K-NS/G 0 33 66B2A2-K-NS/G 50 78 88 B2A2-K-NS/G 100 89 89 B2A2-K-NS/G 300 80 80

The fusion rate for graft material augmented with each B2A2-K-NS dosewas clearly higher than the autograft fusion rate.

The results clearly demonstrate that the B2A2-K-NS delivery kinetics andquantities are optimized to match the amount of host BMP-2 and toenhance its activity in this bony repair indication.

Example 10 Excipients Added to the Peptide as Bulking Agents

Excipients were added to the peptide as bulking agents, stabilizers, toprevent non-specific binding, etc. These excipients which did notinterfere with peptide binding to the granules include the following:

(1) 0.9% saline (no excipients)

(2) 5.5% dextrose

(3) 5.5% dextrose+0.05% Tween 20

(4) 2% mannitol+0.1% dextrose+0.05% polysorbate 80

(5) 5.5% dextrose

(6)₄% mannitol+10 mM glycine

The resulting formulations were bound to 60:40 (HA:TCP) (pH 8.8) and20:80 (HA:TCP) granules. All excipient containing formulations resultedin improved peptide binding to both materials as monitored by HPLC orcolorimetric techniques as compared to formulations, which lackedexcipients.

Example 11 Use of B2A2-K-NS/Ceramic Granules to Augment InstrumentedInterbody Spinal Fusion in Sheep

B2A2-K-NS/ceramic granules (B2A2-K-NS/G) with or without 1:1 v/vmorcelized ilicac crest bone where surgically implanted in the lumbarspines of sheep at L2-L3 and L4-L5 in Tetris® PEEK cages (SignusMedical, LLC). 100% morselized cancellous iliac autograft was used as apositive control material. For the experimental treatments, 50% of theappropriate treatment device and 50% morselized iliac cancellous graftwere implanted. Treatments were performed according to a pre-definedrandomization table and strictly according to the cage manufacturer'srecommendations for device implantation. Morselized cancellous iliaccrest was used for all autograft treatments (positive control). Afterfluoroscopic verification of the previously implanted cage, the twooperative levels (L2-L3 and L4-L5) were then instrumented with theSilhouette™ polyaxial pedicle screw and rod system (Zimmer Spine, Inc.)using standard technique for pedicle preparation and screw placement.For wound closures, the muscle, subcutaneous layer, and skin wereapproximated with running and interrupted 1-0 Vicryl sutures and theskin dosed with staples.

At four months for the B2A2-K-NS/G:autograft group and six months forthe B2A2-K-NS/G lacking autograft group, the animals were sacrificed andthe following analyses were performed. Non-destructive biomechanicaltesting evaluated fusion of the motion segment, CT evaluated fusionwithin the cage and between the two endplates. Additionally, the qualityof bone formed within the VBR (which may be protected from stresses) wasassessed by destructive biomechanical testing.

The following definitions for fusion were utilized:

-   -   CT=At least two sagittal slices on the CT images demonstrated        evidence of contiguous bone from endplate to endplate without        signes of radiolucencies. Two of three blinded examiners had to        agree on the fusion status.    -   Non-destructive biomechanical testing=<3° motion in        flexion-extension    -   The following results are for B2A2-K-NS/G that was mixed 1:1        with autograft after 4 months.

TABLE 6 Fusion rate by non-destructive Treatment Fusion rate by CT (%)biomechanical testing Autograft 100 88 B2A2-K-NS/ 63 88 G 0 ugB2A2-K-NS/ 88 88 G 50 ug B2A2-K-NS/ 88 100 G 100 ug B2A2-K-NS/ 88 75 G300 ug B2A2-K-NS/ 75 88 G 600 ugThe destructive testing demonstrated statistically comparable resultsamong all treatments tested (autograft, B2A2-K-NS/G 0 ug, B2A2-K-NS/G 50ug and B2A2-K-NS/G 100 ug) which indicated comparable bone quality amongall groups. For B2A2-K-NS/G 100 ug that did not contain autograft after6 months, the fusion rate was 86% by CT and 100% by non-destructivebiomechanical testing.

The fusion rate for graft material augmented with B2A2-K-NS 2-K-NS dose(50-300) was comparable to the autograft fusion rate and higher than thegraft material without the B2A2-K-NS coating. The results clearlydemonstrate that the B2A2-K-NS delivery kinetics and quantities areoptimized to match the amount of host BMP-2 and to enhance its activityin this bony repair indication.

Example 12 Use of B2A2-K-NS/Granules with Exogenous Recombinant BMP-2 toInduce Ectopic Bone

Sterile ceramic granules (Berkeley Advanced Biomaterials, Inc.; 60:40(HA:TCP)) where coated by immersing them for 30 minutes in a solutioncontaining B2A2-K-NS (0-, 2.5-, 10-, 25 μg/ml). Thereafter, the solutionwas removed by aspiration and the granules allowed to air dry. Thecoated granules were then placed in sterile gelatin capsules.Immediately before subcutaneous implant in rats, the capsules wereinjected with a collagen-based gel (GFR-Matrigel) containing 1 μgrecombinant BMP-2. The capsules were placed subcutaneously in youngadult rats on the flanks in pre-prepared pouches and the surgical siteclosed with surgical clips. After 30 days, the implants were surgicallyremoved, weighed, and palpated to assess hardness. The explants thenfixed with multiple changes into 70% ethanol. The explants coated with25 μg B2A2-K-NS had a significantly higher weight than controls (noBMP-2, no B2A2-K-NS), and tended (p=0.11) to have a higher weight thanthose containing BMP-2 but no B2A2-K-NS. All implants coated with 25 μgB2A2-K-NS were judged to be palpably hardened (4/4) whereas implantscontaining BMP-2 but no B2A2-K-NS had less hardening (1/4). Implantscoated with 2.5 or 10 μg B2A2-K-NS were intermediate (3/4). Histologicalexamination of the implants following staining with hemotoxylin andeosin revealed new bone formation consistent with the palpation results.All implants coated with 25 μg B2A2-K-NS had histologically identifiablenew bone (4/4), whereas implants containing BMP-2 but no B2A2-K-NS hadless (1/4).

The present invention has been described in terms of preferredembodiments, however, it will be appreciated that various modificationsand improvements may be made to the described embodiments withoutdeparting from the scope of the invention. Variations and modificationsof the present invention will be obvious to those skilled in the art andis intended to cover in the appended claims all such modifications andequivalents. The entire disclosures of all references, applications,patents, and publications cited above are hereby incorporated byreference.

What is claimed is:
 1. A composition comprising: a synthetic growthfactor analogue having the structure

wherein Hx is 6-aminohexanoic acid, and an osteoconductive materialcomprising 60-20 wt % hydroxyapatite and 80-40 wt % tricalciumphosphate; wherein the synthetic growth factor analogue is bound to andcan be released from the osteoconductive material.
 2. The composition ofclaim 1, wherein the osteoconductive material comprises 60 wt %hydroxyapatite and 40 wt % tricalcium phosphate.
 3. The composition ofclaim 1, wherein the osteoconductive material comprises 20 wt %hydroxyapatite and 80 wt % tricalcium phosphate.
 4. The composition ofclaim 1, wherein the osteoconductive material is formed into granules.5. The composition of claim 1, wherein the composition further comprisesmannitol.
 6. The composition of claim 1, wherein the composition furthercomprises glycine.
 7. The composition of claim 1, wherein thecomposition further comprises mannitol and glycine.
 8. The compositionof claim 1, wherein the composition further comprises mannitol; theosteoconductive material comprises 20 wt % hydroxyapatite and 80 wt %tricalcium phosphate; and the osteoconductive material is formed intogranules.
 9. A kit comprising: a vial containing a synthetic growthfactor analogue having the structure

wherein Hx is 6-aminohexanoic acid, and a separate container containingan osteoconductive material comprising 60-20 wt % hydroxyapatite and80-40 wt % tricalcium phosphate.
 10. The kit of claim 9, wherein theosteoconductive material comprises 60 wt % hydroxyapatite and 40 wt %tricalcium phosphate.
 11. The kit of claim 9, wherein theosteoconductive material comprises 20 wt % hydroxyapatite and 80 wt %tricalcium phosphate.
 12. The kit of claim 9, wherein theosteoconductive material is formed into granules.
 13. The kit of claim9, wherein the vial further contains mannitol.
 14. The kit of claim 9,wherein the vial further contains glycine.
 15. The kit of claim 9,wherein the vial further contains mannitol and glycine.
 16. The kit ofclaim 9, wherein the vial further contains mannitol; the osteoconductivematerial comprises 20 wt % hydroxyapatite and 80 wt % tricalciumphosphate; and the osteoconductive material is formed into granules.